Recently, biosensors have become a practical alternative to complex and expensive analytical instruments used in healthcare, agriculture and environmental monitoring. Among several currently used detection technologies such as optical, acoustic and piezoelectric, electrochemical sensors feature prominently due to their simplicity, specificity and high performance. Electrochemical blood glucose sensors are the most commercially successful biosensors accounting for 60% of global biosensor market. The sensors are based on amperometric monitoring of glucose oxidation by recombinant glucose dehydrogenase (GDH). We have developed biosensors by engineering of GDH through insertion and splitting strategies. The sensors can be adopted for the detection of Calcium ion, immunosuppressant drugs, salivary a-amylase protein or protease activity of thrombin and Factor Xa. We expect that the sensor architectures could be expanded to the detection of other biochemical activities, posttranslational modifications, nucleic acids and inorganic molecules. And we also expected the sensors can be used for the developing of Point-of Care diagnostics. Particularly, clinic testing of our first Point-of-Care device developed for Calcium detection for human blood, saliva and urine samples have been proceeded recently.